There is currently no prophylactic vaccine in clinical use for Epstein-Barr virus (EBV). EBV has been strongly implicated in the etiology of Burkitt's lymphoma, Hodgkin lymphoma, nasopharyngeal carcinoma, gastric adenocarcinoma, post-transplantation lymphoproliferative disease, and infectious mononucleosis. Collectively these diseases cause substantial global morbidity and mortality. Extensive research indicates a critical role for four EBV proteins in mediating viral binding and entry into host target cells (B cells and epithelial cells), i.e. EBV gp350, gH/gL, and gB. The rabbit elicits robust neutralizing antibody responses in response to EBV proteins and provides adequate quantities of sera for extensive EBV neutralization studies. Established assays are also available for initial in vitro screening of sera from immunized rabbits for neutralizing antibody titers using target B cells and epithelial cells. We produced multimeric recombinant EBV gp350, gH/gL, and gB and their corresponding monomeric proteins, using DNA constructs stably transfected in CHO cells. In preliminary studies we demonstrated that rabbits immunized and boosted s.c. with either EBV gp350 or EBV gH/gL in alum elicited high-titer serum IgG anti-gp350 and IgG anti-gH/gL antibody responses. Immune sera from both groups were effective in specifically inhibiting EBV infection of primary human peripheral blood B cells. In this proposal we plan to substantially extend these preliminary studies through immunization of rabbits with EBV gp350, gH/gL, and gB in the presence of alum + CpG-ODN and determining the ability of immune sera to 1) inhibit EBV infection of primary human peripheral blood B cell and the Raji human Burkitt lymphoma B cell line, 2) prevent EBV infection of primary human tonsillar epithelial cells, using a B cell- mediate EBV transfer infection model, and a CD21-expressing epithelial cell line (SVKCR2), and 3) confer passive protection against EBV infection in humanized (h)NOG mice. Collectively, these experiments will take a multi-faceted approach to determine the potential additive or synergistic EBV- neutralizing capacity of antibodies in response to four key protein targets involved in primary EBV infection, with potential for informing the development of a sterilizing prophylactic EBV vaccine.